This past year we have made significant discoveries for each of these objectives summarized below. Identify human viruses (in addition to PV, CVB3 and rhinovirus) that traffic by bloc transmission both in vitro and in vivo. (Marianita Santiana, Sourish Ghosh, Brian Ho) We have discovered that two important human enteric viruses, rotavirus and norovirus, both exit cells as populations using vesicles. We have found that rotavirus vesicles are derived from the plasma membrane and norovirus vesicles are derived from multivesicular bodies. Furthermore, we developed methods to look for whether these two viruses can be transmitted in vivo inside vesicles. With help from our collaborators (Kim Green, Wendy Henderson, Linda Saif) we obtained rotavirus and norovirus infected fecal samples and demonstrated that both viruses are present inside phosphatidylserine-enriched vesicles in fecal samples. We also obtained fecal samples from Astrovirus and Parvovirus infected individuals (thorugh our collaborator David Wang) and developed methods to isolate putative astro or parvo virus carrying fecal vesicles. We are currently analyzing the contents of the vesicles pulled down to determine if they carry these viruses. In addition we are pulling down fecal vesicles from unknown samples and doing RNA and DNA sequencing to identify additional viruses that are being trafficked inside vesicles in feces. Determine the mechanisms by which identified viruses are trafficked out of cells as populations both in vitro and in vivo. (Yael Mutsafi Benhalevy, Teegan Dellibovi-Ragheb) We have identified some of the autophagy machinery being exploited for poliovirus exit in secretory autophagosomes as well as a potential viral protein sequence that is being targeted by the host autophagy machinery to capture poliovirus particles. We have also discovered a novel pathway for coronavirus exit from cells. Contrary to literature that states that these viruses ( SARS, MERS etc) leave cells via the secretory pathway, we have found instead that these viruses are secreted from cells by the fusion with the plasma membrane of an endoplasmic reticulum- late endosome hybrid compartment. Determine the impact, in terms of viral yield, virulence and viral genetic diversity, of viral population carrying vesicles in inter-organismal transmission. (Sourish Ghosh, Marianita Santiana) This past year we have developed an in vivo mouse model system to interrogate the role of vesicles in rotavirus transmission. We have shown that vesicles carrying murine rotavirus are stable and can transmit infection through the oral-fecal route (much like in humans). Moreover we have found that this type of transmission, compared to free rotavirus transmission, results in significantly more severe clinical symptoms, earlier clinical symptom onset, longer disease period and much greater transmission to littermates. We have also begun to examine which tissues are preferentially targeted by viruses carrying populations, what their targeting may mean for the clinical severity observed. We will also begin examining the viral genome diversity in an organism that has been infected by populations as opposed to free particles. Determine the differences, if any, in the host immune responses (innate and adaptive) when infected by bloc transmission versus single particle transmission. (Ying-Han Chen, Sourish Ghosh, Yakey Yaffe) We have found profound differences in the innate and adaptive immune response of cells and organisms respectively to infection by bloc transmission. In summary when cells are infected with high multiplicities of viral genomes we find that they can no longer distinguish among entering non-self RNA and self RNA molecules, leading to an overall suppression of the innate immune response. This is a completely unexpected finding and upends much of what we know about innate immune responses and self/non-self RNA recognition. In addition, we find profound differences in the adaptive immune responses, specifically the mucosal immune response. This coming FY we will investigate if the innate immune behavior (suppression) is linked to the mucosal immune behavior (suppression).